Biologically active polypeptides or proteins which are attached to insoluble carrier materials, such as polymeric particles, have been used in a variety of ways. For example, the diagnosis of pathological or other conditions in human beings and animals is often carried out using immunological principles for the detection of an immunologically reactive species, for example antibodies or an antigen, in the body fluids of the person or animal. An antigen is generally known as a foreign substance, such as a drug, hapten, toxin, lectin, glycoprotein, polysaccharide, glycolipid, polypeptide or protein which, when introduced into the body, causes the production of certain soluble proteins known as antibodies.
Other proteins (such as enzymes or avidin) or biotin, have been covalently linked to various carrier materials for use in affinity chromatography, enzymatic reactions, analytical procedures, specific binding reactions and immunoassays. Among useful carrier materials are sheep and human erythrocytes, bacterial cells, latex particles, resinous particles and finely divided diazotized amino cellulose. For example, carrier particles prepared from sparingly water-soluble monomers (such as epoxy group-containing monomers) in the absence of emulsifiers are described in U.S. Pat. No. 4,15,700 (issued Nov. 15, 1983 to Batz et al).
The antigen-antibody reaction is the basis or all immunological test methods. The normal body response to a foreign substance has led to the development of a number of techniques which are used to diagnose various diseases, disorders and physiological conditions. Other specific binding reactions can occur between a ligand and corresponding receptor compounds. For example, avidin or a derivative thereof specifically reacts with biotin or a derivative thereof. Other specific binding reactions occur between enzymes and their substrate analogs, inhibitors or cofactors, lectins and sugars (including mono- , oligo- and polysaccharides and glycoproteins), nucleic acids and complementary base sequences, histones and binding proteins, and hormones and their receptors. In a general sense, one component of the reaction can be defined as the ligand while the corresponding component which reacts with it is considered the receptor.
In vitro tests for the presence of a suspected protein (for example, an antigen or antibody) in a biological sample are carried out by adding the immunological counterpart to the biological sample. If the suspected substance is present, the resulting antigen-antibody reaction can be demonstrated by precipitation of the antigen-antibody complex. This reaction complex is generally difficult to detect visually. For this reason, either antibodies or antigens are often bound to insoluble particles, for example polymer latex particles, so that when the complex is formed, it is readily detectable from the resulting agglutination either by observing the presence of clumping or by a detectable tracer associated with the particles. Agglutination is characterized by the clumping of particles from a suspension of particles. Further details of known agglutination methods are provided in U.S. Pat. No. 4,419,453 (issued Dec. 6, 1983 to Dorman et al) and U.S. Pat. No. 4,459,361 (issued July 10, 1984 to Gefter).
Immunoreactive reagents are described in U.S. Pat. No. 4,259,313 (issued Mar. 31, 1981 to Frank et al). These reagents are composed of a polymer or mixture of polymers in particulate form having distributed throughout the particles a fluorescent rare earth chelate which renders the reagents detectable using suitable equipment. Attached to the particles are molecules of an immunological species such as an antibody or antigen. The resulting reagent can be used in immunoassays. However, fluorometric equipment is required with these reagents. There are many instances where such equipment is not available or practical. For example, it would be useful to have immunoreactive reagents which can be detected readily by a user without the need for detection equipment either in a consumer's home or in a doctor's office. Such reagents would desirably be detectable within a few minutes visually with the unaided eye.
As a result, it has been proposed to incorporate various dyes or color-forming materials within such reagents by dispersing them within the polymeric particle as described, for example, in E.P. Publication 227,173. While this makes the reagents readily detectable, surprisingly it has been found that dye molecules on the surface of the particles often interfere with immunological reactions, such as those required for agglutination assays. Other materials on the surface of such particles, such as surfactants or stabilizers can also similarly interfere with the reactions.
U.S. Pat. No. 4,401,765 (issued Aug. 30, 1983 to Craig et al) describes a reagent which is a core/shell polymer having an immunological species attached thereto. These reagents contain no tracer material, and are detected in the assay through light scattering techniques which require sophisticated equipment that would be available only in a clinical chemistry laboratory. Even the simplest technique involving the measurement of turbidity requires the use of a spectrophotometer (see Col. 4, lines 1-56). These techniques clearly would not be useful for assays which are to be carried out at home, in doctors' offices and other places where equipment is limited or impractical. Hence, there is a need for a readily detectable immunoreactive reagent which can be easily detected without complicated equipment and procedures and which avoids the interference caused by materials on the surface of particles.